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Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans

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Abstract

Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43–2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60–2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44–2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38–3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation.

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References

  1. Storey KB (1996) Metabolic adaptations supporting anoxia tolerance in reptiles: recent advances. Comp Biochem Physiol B Biochem Mol Biol 113(1):23–35

    Article  PubMed  CAS  Google Scholar 

  2. Storey KB (2007) Anoxia tolerance in turtles: metabolic regulation and gene expression. Comp Biochem Physiol A Mol Integr Physiol 147(2):263–276

    Article  PubMed  Google Scholar 

  3. Ultsch GR (2006) The ecology of overwintering among turtles: where turtles overwinter and its consequences. Biol Rev Camb Philos Soc 81(3):339–367

    Article  PubMed  Google Scholar 

  4. Herbert CV, Jackson DC (1985) Temperature effects on the response to prolonged submergence in the turtle Chrysemys picta bellii. II. Metabolic rate, blood acid-base and ionic changes, and cardiovascular function in aerated and anoxic water. Physiol Zool 58:670–681

    Google Scholar 

  5. Hochachka PW, Lutz PL (2001) Mechanism, origin, and evolution of anoxia tolerance in animals. Comp Biochem Physiol B Biochem Mol Biol 130B:435–459

    Article  CAS  Google Scholar 

  6. Jackson DC (1968) Metabolic depression and oxygen depletion in the diving turtle. J Appl Physiol 24:503–509

    PubMed  CAS  Google Scholar 

  7. Krivoruchko A, Storey KB (2010) Forever young: mechanisms of natural anoxia tolerance and potential links to longevity. Oxid Med Cell Longev 3(3):186–198

    Article  PubMed  Google Scholar 

  8. Krivoruchko A, Storey KB (2010) Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans. J Comp Physiol B 180(3):403–414

    Article  PubMed  CAS  Google Scholar 

  9. Prentice HM, Milton SL, Scheurle D, Lutz PL (2004) The upregulation of cognate and inducible heat shock proteins in the anoxic turtle brain. J Cereb Blood Flow Metab 24(7):826–828

    Article  PubMed  CAS  Google Scholar 

  10. Schröder M (2008) Endoplasmic reticulum stress responses. Cell Mol Life Sci 65(6):862–894

    Article  PubMed  Google Scholar 

  11. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmicreticulum-resident kinase. Nature 397:271–274

    Article  PubMed  CAS  Google Scholar 

  12. Shi Y, An J, Liang J, Hayes S, Sandusky GE, Stramm LE, Yang NN (1999) Characterization of a mutant pancreatic eIF-2a kinase, PEK, and co-localization with somatostatin in islet delta cells. J Biol Chem 274:5723–5730

    Article  PubMed  CAS  Google Scholar 

  13. Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L, Wek RC (1998) Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol 18:7499–7509

    PubMed  CAS  Google Scholar 

  14. Dorner AJ, Wasley LC, Kaufman RJ (1992) Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells. EMBO J 11:1563–1571

    PubMed  CAS  Google Scholar 

  15. Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J (1988) The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332:462–464

    Article  PubMed  CAS  Google Scholar 

  16. Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2:379–384

    Article  PubMed  CAS  Google Scholar 

  17. Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K, Mori K (2006) Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol 172:383–393

    Article  PubMed  CAS  Google Scholar 

  18. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258

    Article  PubMed  CAS  Google Scholar 

  19. Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, Mori K (2003) A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 4:265–271

    Article  PubMed  CAS  Google Scholar 

  20. Sidrauski C, Walter P (1997) The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90(6):1031–1039

    Article  PubMed  CAS  Google Scholar 

  21. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881–891

    Article  PubMed  CAS  Google Scholar 

  22. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D (2000) Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5:897–904

    Article  PubMed  CAS  Google Scholar 

  23. Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K (2000) ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol Cell Biol 20:6755–6767

    Article  PubMed  CAS  Google Scholar 

  24. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11:619–633

    Article  PubMed  CAS  Google Scholar 

  25. Ron D (2002) Translational control in the endoplasmic reticulum stress response. J Clin Invest 110:1381–1388

    Google Scholar 

  26. Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T, Bonner-Weir S, Kaufman RJ (2001) Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell 7:1165–1176

    Article  PubMed  CAS  Google Scholar 

  27. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  PubMed  CAS  Google Scholar 

  28. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5):1475–1489

    Article  PubMed  CAS  Google Scholar 

  29. Krivoruchko A, Storey KB (2010) Molecular mechanisms of turtle anoxia tolerance: a role for NF-kappaB. Gene 450(1–2):63–69

    Article  PubMed  CAS  Google Scholar 

  30. Krivoruchko A, Storey KB (2010) Activation of antioxidant defenses in response to freezing in freeze-tolerant painted turtle hatchlings. Biochim Biophys Acta 1800:662–668

    Article  PubMed  CAS  Google Scholar 

  31. Wek RC, Cavener DR (2007) Translational control and the unfolded protein response. Antioxid Redox Signal 9(12):2357–2371

    Article  PubMed  CAS  Google Scholar 

  32. Schröder M, Kaufman RJ (2005) The mammalian unfolded protein response. Annu Rev Biochem 74:739–789

    Article  PubMed  Google Scholar 

  33. Schröder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569(1–2):29–63

    PubMed  Google Scholar 

  34. Jackson DC, Ultsch GR (2010) Physiology of hibernation under the ice by turtles and frogs. J Exp Zool A Ecol Genet Physiol 313(6):311–327. doi:10.1002/jez.603

    Article  PubMed  Google Scholar 

  35. Warren DE, Jackson DC (2008) Lactate metabolism in anoxic turtles: an integrative review. J Comp Physiol B 178(2):133–148. doi:10.1007/s00360-007-0212-1

    Article  PubMed  CAS  Google Scholar 

  36. Jackson DC (2004) Acid-base balance during hypoxic hypometabolism: selected vertebrate strategies. Respir Physiol Neurobiol 141(3):273–283. doi:10.1016/j.resp200401009

    Article  PubMed  CAS  Google Scholar 

  37. Fels DR, Koumenis C (2006) The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol Ther 5(7):723–728

    Article  PubMed  CAS  Google Scholar 

  38. Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8(7):519–529

    Article  PubMed  CAS  Google Scholar 

  39. Ameri K, Hammond EM, Culmsee C, Raida M, Katschinski DM, Wenger RH, Wagner E, Davis RJ, Hai T, Denko N, Harris AL (2007) Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway. Oncogene 26(2):284–289

    Article  PubMed  CAS  Google Scholar 

  40. Vatten KM, Wek RC (2004) Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA 101(31):11269–11274

    Article  Google Scholar 

  41. Hai T (2006) The ATF transcription factors in cellular adaptive responses. In: Ma J (ed) Gene expression and regulation. Higher Education Press, Beijing, pp 322–333

    Google Scholar 

  42. Lu D, Wolfgang CD, Hai T (2006) Activating transcription factor 3, a stress-inducible gene, suppresses Ras-stimulated tumorigenesis. J Biol Chem 281(15):10473–10481

    Article  PubMed  CAS  Google Scholar 

  43. Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. J Cell Biol 153(5):1011–1022

    Article  PubMed  CAS  Google Scholar 

  44. Connor JH, Weiser DC, Li S, Hallenbeck JM, Shenolikar S (2001) Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1. Mol Cell Biol 21(20):6841–6850

    Article  PubMed  CAS  Google Scholar 

  45. Lee AS (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26(8):504–510

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Jan Storey for editorial review of this manuscript. This research was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs program.

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  1. Anastasia Krivoruchko

    Present address: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden

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  1. Department of Biology, Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada

    Anastasia Krivoruchko & Kenneth B. Storey

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  2. Kenneth B. Storey
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Correspondence to Anastasia Krivoruchko.

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Krivoruchko, A., Storey, K.B. Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans . Mol Cell Biochem 374, 91–103 (2013). https://doi.org/10.1007/s11010-012-1508-3

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